Biomod/2011/TUM/TNT/Results

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<h2>New practical methods and theories will be needed</h2>
<h2>New practical methods and theories will be needed</h2>
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Therefore, FRET measurements would be an appropriate method. Although we cannot present final results for FRET analyses, first single molecule analyses can be provided, and the fluorescence tracking approach already displays some tendencies. For an optimization of the FRET studies, the origami structure needs some slight imrpovements. For this, we have layed a thorough fundament not only of experimental results, but also lots of theoretical considerations, which can explain flexibility and correlate observable with unobservable deformations.  
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Therefore, FRET measurements would be an appropriate method. Although we cannot present final results for FRET analyses, first single molecule analyses can be provided, and the fluorescence tracking approach already displays some tendencies. For an optimization of the FRET studies, the origami structure needs some slight improvements. For this, we have laid a thorough fundament not only of experimental results, but also lots of theoretical considerations, which can explain flexibility and correlate observable (via TEM and / or fluorescence measurements: distances, angles) with unobservable (twists) structural changes. <br>
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On the experimental side, one could  try to eliminate some uncertainties regarding the applied concentrations. We did some [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Calculation_of_intercalator_concentrations calculations] to determine the fraction of occupied binding sites even at small concentrations, but as mentioned above, binding could be cooperative and for a proper testing of such a behavior, concentrations of bound DNA binders must be checked experimentally. This is very trying due to the small concentrations and the little fraction of compounds bound compared to those free in solution. We suggest to try some radiolabeled DNA binders, of which the bound fraction can be determined from radioassays. <br>
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Using this information, it should be possible to unravel deformation events step by step on even tinier levels. This not only allows for sophisticated understanding of the flexibility of origamis in response to varying triggers, thereby enabling the development of custom-made dynamic structures, but also helps elucidating the mechanic and maybe also mechanistic effects of DNA binders.
<h1>Outlook</h1>
<h1>Outlook</h1>
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For this last goal, we have the vision of a characteristic plot for DNA binders based on the twist and length changes they cause. In the field of bioscience the Ramachandran plot is a nice way to show typical secondary structures of proteins. In this case we would plot twist against length (fig zzz). <br>
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Referring to the relevance of this project accordingly the future goal should be to find a way to characterize DNA binding molecules. In the field of bioscience the Ramachandran plot is a nice way to show typical secondary structures of proteins.  
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In this case we would plot twist against length (fig 1). To do this it would be necessary to test more DNA binding molecules and to design a structure which is more sensitive to measure changes in length. This should be done based on the theoretical background which considers geometrical thoughts and information we gained during this project.
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[[Image:Plotslim.png | 600px|center]]<br>
[[Image:Plotslim.png | 600px|center]]<br>
<div style="text-align: center"><font size="1">fig. 1: Classifying DNA-binders by twist and length change</font size><br></div>
<div style="text-align: center"><font size="1">fig. 1: Classifying DNA-binders by twist and length change</font size><br></div>
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By optimizing the structure based on our results it should be possible to create a device whose conformational changes can be precisely predetermined.
 
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This would permit to use this structure the other way around. Knowing the outcome of conformational changes of DNA origami using a certain concentration of an well known DNA-binder will provide a valuable tool especial in the field of creating functional DNA origami.
 
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Welche Experimente wären noch möglich/ sollten noch gemacht werden, um... zu zeigen. Ferne und nahe Zukunft. Was könnte damit noch untersucht werden (Sequenzspezifische Bindeproteine...)
 
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* Struktur mit asymmetrischem feature um die richtung der projektion bestimmen zu können
 

Revision as of 19:17, 2 November 2011

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